Novel anti-adipogenic effect of CF3-allylated indole in 3T3-L1 cells.

Indole derivatives from various plants are known to have health benefits because of their anti-cancer, anti-oxidant, anti-inflammatory, and anti-tubercular effects. However, their effects on adipogenesis have not been fully elucidated yet. Herein, we show that a newly synthesized indole derivative, CF3-allylated indole, [(E)-1-(pyrimidin- 2-yl)-2-(4,4,4- trifluorobut-2-enyl)-1H-indole], effectively inhibits adipogenesis. We found that CF3-allylated indole inhibited lipid accumulation and suppressed the expression of CCAAT/enhancer-binding protein α (C/EBPα) and peroxisome proliferator activated receptor γ (PPARγ) in 3T3-L1 cells. The inhibitory effect of CF3-allylated indole primarily occurred at the early phase of adipocyte differentiation by increasing intracellular cyclic adenosine monophosphate (cAMP) levels and enhancing protein kinase A (PKA) and adenosine monophosphate-activated protein kinase (AMPK) signaling. Conversely, depletion of PKA or treatment with a protein kinase A inhibitor (H89) reversed such inhibitory effects of CF3-allylated indole on adipogenesis and PPARγ expression. These results suggest that CF3-allylated indole inhibits early stages of adipogenesis by increasing phosphorylation of PKA/AMPK, leading to decreased expression of adipogenic genes in 3T3-L1 cells. These results indicate that CF3-allylated indole has potential for controlling initial adipocyte differentiation in metabolic disorders such as obesity.

Exosome-Mediated Differentiation of Mouse Embryonic Fibroblasts and Exocrine Cells into ß-Like Cells and the Identification of Key miRNAs for Differentiation.

Diabetes is a concerning health malady worldwide. Islet or pancreas transplantation is the only long-term treatment available; however, the scarcity of transplantable tissues hampers this approach. Therefore, new cell sources and differentiation approaches are required. Apart from the genetic- and small molecule-based approaches, exosomes could induce cellular differentiation by means of their cargo, including miRNA. We developed a chemical-based protocol to differentiate mouse embryonic fibroblasts (MEFs) into ß-like cells and employed mouse insulinoma (MIN6)-derived exosomes in the presence or absence of specific small molecules to encourage their differentiation into ß-like cells. The differentiated ß-like cells were functional and expressed pancreatic genes such as Pdx1, Nkx6.1, and insulin 1 and 2. We found that the exosome plus small molecule combination differentiated the MEFs most efficiently. Using miRNA-sequencing, we identified miR-127 and miR-709, and found that individually and in combination, the miRNAs differentiated MEFs into ß-like cells similar to the exosome treatment. We also confirmed that exocrine cells can be differentiated into ß-like cells by exosomes and the exosome-identified miRNAs. A new differentiation approach based on the use of exosome-identified miRNAs could help people afflicted with diabetes.

Carbamazepine Enhances Adipogenesis by Inhibiting Wnt/ß-catenin Expression.

Carbamazepine is a drug that is widely used in the treatment of epilepsy and bipolar disorder. The prevalence of obesity in patients treated with carbamazepine has been frequently reported. However, whether carbamazepine affects adipogenesis, one of the critical steps in the development of obesity, remains unclear. Here, we show that carbamazepine increased the expression levels of peroxisome proliferator-activated receptor γ (PPARγ), CCAAT/enhancer-binding protein ß (C/EBPß), and fatty acid synthase (FASN) in 3T3-L1 cells. Notably, carbamazepine inhibited the expression levels of ß-catenin, a negative regulator of adipogenesis, leading to enhanced adipogenesis. Conversely, ß-catenin overexpression abolished the effect of carbamazepine on adipogenic gene expression. However, depletion of ß-catenin further enhanced PPARγ expression. In addition, carbamazepine reduced ß-catenin expression by lowering the levels of phospho-low density lipoprotein receptor-related protein 6 (p-LRP6) and phospho-glycogen synthase kinase 3ß (p-GSK3ß) in Wnt/ß-catenin signaling. Moreover, carbamazepine reduced Wnt mRNA expression and decreased the promoter activities of TCF, the target of ß-catenin during adipogenesis. These results suggest that carbamazepine enhances adipogenesis by suppressing Wnt/ß-catenin expression, indicating its potential effects on obesity-related metabolism.

mTOR controls ChREBP transcriptional activity and pancreatic ß cell survival under diabetic stress.

Impaired nutrient sensing and dysregulated glucose homeostasis are common in diabetes. However, how nutrient-sensitive signaling components control glucose homeostasis and ß cell survival under diabetic stress is not well understood. Here, we show that mice lacking the core nutrient-sensitive signaling component mammalian target of rapamycin (mTOR) in ß cells exhibit reduced ß cell mass and smaller islets. mTOR deficiency leads to a severe reduction in ß cell survival and increased mitochondrial oxidative stress in chemical-induced diabetes. Mechanistically, we find that mTOR associates with the carbohydrate-response element-binding protein (ChREBP)-Max-like protein complex and inhibits its transcriptional activity, leading to decreased expression of thioredoxin-interacting protein (TXNIP), a potent inducer of ß cell death and oxidative stress. Consistent with this, the levels of TXNIP and ChREBP were highly elevated in human diabetic islets and mTOR-deficient mouse islets. Thus, our results suggest that a nutrient-sensitive mTOR-regulated transcriptional network could be a novel target to improve ß cell survival and glucose homeostasis in diabetes.

AMP-activated protein kinase mediates the antioxidant effects of resveratrol through regulation of the transcription factor FoxO1.

Numerous physiological functions are controlled by redox-responsive signaling pathways. Disruption of redox balance by oxidative stress is recognized as a major cause of many pathological conditions, including aging, highlighting the importance of investigating how antioxidants maintain redox homeostasis. AMP-activated protein kinase (AMPK) is activated in response to cellular conditions that accompany energy depletion and plays a central role in the regulation of energy homeostasis, tumorigenesis and longevity. Recently, several antioxidants have been reported to activate AMPK, although the mechanisms by which AMPK acts to adjust the levels of cellular reactive oxygen species are not fully characterized. In the present study, we investigated the role of AMPK in mediating resveratrol-induced antioxidant effects and the molecular mechanisms underlying its actions. We demonstrate that AMPK activity plays an indispensable role in the operation of the ROS defense system by inducing the expression of the antioxidant enzymes, manganese superoxide dismutase and catalase, in response to resveratrol or the AMPK agonist 5-aminoimidazole-4-carboxamide-1-ß-d-ribonucleotide. In addition, we identified the mechanism involved in the antioxidant function of AMPK, demonstrating that AMPK directly phosphorylates human FoxO1 (forkhead box O1) at Thr(649) in vitro and increases FoxO1-dependent transcription of manganese superoxide dismutase and catalase. Mutagenesis studies showed that this AMPK-mediated phosphorylation of FoxO1 is critical for FoxO1 stability and nuclear localization, establishing the molecular basis for the induction of FoxO1 transcriptional activity. Our results reveal a novel FoxO1-dependent mechanism by which AMPK controls the expression of antioxidant enzymes and suggest that AMPK has an important role in maintaining redox homeostasis.

AMP-activated protein kinase α2 and E2F1 transcription factor mediate doxorubicin-induced cytotoxicity by forming a positive signal loop in mouse embryonic fibroblasts and non-carcinoma cells.

Doxorubicin is one of the most widely used anti-cancer drugs, but its clinical application is compromised by severe adverse effects in different organs including cardiotoxicity. In the present study we explored mechanisms of doxorubicin-induced cytotoxicity by revealing a novel role for the AMP-activated protein kinase α2 (AMPKα2) in mouse embryonic fibroblasts (MEFs). Doxorubicin robustly induced the expression of AMPKα2 in MEFs but slightly reduced AMPKα1 expression. Our data support the previous notion that AMPKα1 harbors survival properties under doxorubicin treatment. In contrast, analyses of Ampkα2(-/-) MEFs, gene knockdown of AMPKα2 by shRNA, and inhibition of AMPKα2 activity with an AMPK inhibitor indicated that AMPKα2 functions as a pro-apoptotic molecule under doxorubicin treatment. Doxorubicin induced AMPKα2 at the transcription level via E2F1, a transcription factor that regulates apoptosis in response to DNA damage. E2F1 directly transactivated the Ampkα2 gene promoter. In turn, AMPKα2 significantly contributed to stabilization and activation of E2F1 by doxorubicin, forming a positive signal amplification loop. AMPKα2 directly interacted with and phosphorylated E2F1. This signal loop was also detected in H9c2, C2C12, and ECV (human epithelial cells) cells as well as mouse liver under doxorubicin treatment. Resveratrol, which has been suggested to attenuate doxorubicin-induced cytotoxicity, significantly blocked induction of AMPKα2 and E2F1 by doxorubicin, leading to protection of these cells. This signal loop appears to be non-carcinoma-specific because AMPKα2 was not induced by doxorubicin in five different tested cancer cell lines. These results suggest that AMPKα2 may serve as a novel target for alleviating the cytotoxicity of doxorubicin.